Autonomic activation mediates the majority of increase of glucagon secretion during insulin-induced hypoglycemia in several species, including dogs, mice, and rats. However, the role of the autonomic nervous system in mediating this response in humans is controversial. Autonomic mediation of glucagon secretion during hypoglycemia has not been previously investigated in nonhuman primates. To determine the autonomic contribution to hypoglycemia-induced glucagon secretion in nonhuman primates, we compared glucagon responses in chronically catheterized conscious rhesus monkeys with and without the ganglionic blocking agent, trimethaphan, or with combined muscarinic and adrenergic receptor blockade, with atropine, propranolol, and tolazoline. Insulin-induced hypoglycemia activated parasympathetic nerves to the pancreas as assessed by increased plasma pancreatic polypeptide levels, produced sympathoadrenal activation as assessed by elevations of plasma norepinephrine and epinephrine, and increased plasma immunoreactive glucagon (IRG). Nicotinic ganglionic blockade with trimethaphan prevented parasympathetic and sympathoadrenal activation during hypoglycemia and inhibited the irg response by 70%. Combined muscarinic and adrenergic receptor blockade inhibited the irg response similarly to ganglionic blockade with trimethaphan. These results demonstrate that autonomic activation mediates the majority of the glucagon response to insulin-induced hypoglycemia in rhesus monkeys. The increase of glucagon secretion is primarily due to stimulation of classical muscarinic and adrenergic receptors and therefore unlikely to involve nonclassical neuropeptidergic activation. Nonhuman primates may be valuable as a model for the investigation of an autonomic component to the impairment of hypoglycemia-induced glucagon secretion in diabetes mellitus.